Once acetylcholinesterase (AChE) reacts with organophosphorous compounds (OPCs) developed for chemical warfare, there is very little anyone can do in case of a massive real-life crisis. Part of the issue is that broad-spectrum antidotes to reverse the effects of OPC exposure have yet to be developed. We have recently discovered that amodiaquine, a well-established anti-malarial drug, is a good candidate for such an antidote: it acts as a reactivator of acetylcholinesterase (AChE) from adducts with organophosphorous compounds (OPCs) via a yet undefined, but not oxime-based, mechanism. In this proposal we will test the hypothesis that lipophilic amodiaquine is suitable for use in viv as a post-exposure treatment of organophosphorous poisoning. We will also study the mechanism of reactivation and demonstrate that we can use amodiaquine and related compounds as scaffolds to achieve reactivation under more optimal conditions. We will pursue three aims: In Aim 1 we will provide a detailed biochemical and mechanistic characterization of the interactions of amodiaquine and its close structural analogs with AChE and different adducts that AChE forms with organophosphorous compounds. In Aim 2 we will obtain crystal structures of amodiaquine and its selected analogs with native and OPC inhibited AChE, in order to facilitate rational design of efficient reactivators. In Aim 3 we will study the ability of amodiaquine to reverse the effects of organophosphorous compounds in vivo, particularly focusing on reactivation of AChE in brain. The results of our experiments will firmly establish amodiaquine as the first member of a new class of reactivators of AChE, enabling pre-clinical studies of post-OPC-exposure treatment. Our mechanistic studies will also help us to generate additional analogs suitable for chronic administration (chemoprophylaxis) as well. PUBLIC HEALTH RELEVANCE: The successful medical management of organophosphate poisoning requires completely new leads that can enable reversal of the effects of exposure to nerve agents and pesticides. We propose that a well- established and broadly used anti-malarial drug represents such a lead, being the first member of a new class of reactivators of acetylcholinesterase (AChE) from its complexes with organophosphorous compounds. We describe studies that will (i) demonstrate in vivo activity of this drug on inhibited AChE in brain and (ii) provide mechanistic and structural insights into the reactivation process, enabling rational design of even better agents.